HIV clade C, endemic in sub-Saharan Africa and the Indian subcontinent, is responsible for nearly half of all global HIV infections, and most large-scale HIV vaccine trials are targeted to clade C regions. Unfortunately, the development and trialing of candidate HIV vaccines brings with it the significant risk of vaccine-induced HIV seropositivity (VISP). VISP is defined as the presence of circulating antibodies to HIV vaccine antigens which cross-react with homologous antigens used in conventional HIV immunoassays, leading to a false positive result. In recent HVTN-sponsored HIV vaccine trials, an overall 41.7% of HIV vaccine recipients exhibited VISP. False positive test results can have tragic personal consequences and, from a public health perspective, the anticipated high rate of VISP would make it impossible to monitor effectiveness of an HIV vaccine trial or vaccination campaign, or to correctly diagnose and treat infected individuals. RNA testing, central to current VISP testing algorithms, fails to detect 2-3% of established HIV infections, and its high cost is a burden to vaccine trial budgets and a barrier to use by resource-limited health providers. The goal of the Phase I project is to demonstrate feasibility of a clade C-specific HIV-1 immunoassay (HIV Selectest) capable of distinguishing true HIV infection from VISP with high sensitivity and specificity. The rationale behind a clade-specific assay is that it will enable greater detection sensitivity by increasing the number of clade C HIV peptide sequence variants in the space available in the assay well. The project objectives will be achieved through two innovative approaches: 1) clade-specific targeting of assay antigens to improve detection sensitivity and 2) the novel use of database-derived peptide design tools to achieve optimal coverage of HIV variants. Preliminary data have demonstrated that significant gains in detection sensitivity for challenging samples, early infections and overall sample populations are possible through these approaches. Progress will be assessed by ELISA testing of individual antigenic peptides and peptide mixes vs well-characterized serum panels. Through collaborators in research and blood screening laboratories we will acquire panels of sera from HIV vaccine trials in clade C regions as well as from well-characterized clade C HIV-positive and negative donors. In Phase I we expect to demonstrate feasibility of a clade C HIV Selectest assay capable of distinguishing true HIV infection from VISP, with sensitivity and specificity approaching those of licensed HIV immunoassays when tested on the same target serum panels. In Phase II we will further optimize and scale up the assay and perform prospective and retrospective clinical studies aimed at FDA clearance for use of the Clade C HIV Selectest assay as a cost effective solution to resolve false positives due to VISP in HIV vaccine recipients.